Detection of Antigen-Specific B Cells in Patients with Pemphigus Vulgaris by Enzyme-Linked Immunospot Assay: Requirement of T Cell Collaboration for Autoantibody Production

Abstract

Patients with pemphigus vulgaris have circulating IgG autoantibodies against desmoglein 3, which inhibit cell-cell adhesion of keratinocytes and cause blister formation in the skin and mucous membrane. To examine cellular mechanisms underlying the autoantibody production in pemphigus vulgaris patients, we have successfully developed an enzyme-linked immunospot assay which was able to detect desmoglein 3-specific autoimmune B cells quantitatively. Circulating B cells producing anti-desmoglein 3 antibodies were detected exclusively in three patients with severe disease (1.3-2.3/105 peripheral blood mononuclear cells), but not in 10 patients with mild disease or in remission or in seven healthy individuals. When this enzyme-linked immunospot assay was combined with in vitro stimulation of peripheral blood mononuclear cells with pokeweed mitogen and recombinant-desmoglein 3, we could detect circulating desmoglein 3-specific memory B cells in nine of 14 patients (6.3-84. 0/105 peripheral blood mononuclear cells), but in none of 10 healthy individuals. We further analyzed the role of CD4 + T cells in promoting anti-desmoglein 3 antibody production. The in vitro anti-desmoglein 3 antibody production was abolished when CD4 + cells were depleted or when anti-HLA-DR or anti-HLA-DQ monoclonal antibody was added to the cultures. Our results demonstrated the quantitative detection of circulating "activated" and "memory" desmoglein 3-specific B cells and suggested the important part of HLA class II-restricted CD4 + T cells in the autoantibody production in pemphigus vulgaris. In addition, the enzyme-linked immunospot assay in combination with in vitro stimulation of B cells could be broadly applied to study mechanisms for autoantibody production in various autoimmune diseases.

Inhibition of the Transcription Factor Foxp3 Converts Desmoglein 3-Specific Type 1 Regulatory T Cells into Th2-Like Cells

“…A critical role for autoreactive T cells in the induction and regulation of Dsg3 autoantibody production has been suggested in a recent study by Nishifuji et al (14). Anti-Dsg3 autoantibodies secreted by autoreactive B cells were detected by ELISPOT assay upon in vitro stimulation of peripheral lymphocytes from PV patients with Dsg3.…”

T Cell Recognition of Desmoglein 3 Peptides in Patients with Pemphigus Vulgaris and Healthy Individuals

“…Recent evidence suggests that these HLA class II alleles are involved in the presentation of Dsg3 peptides to autoreactive T cells in PV patients (19 -22). Autoreactive Th cells are presumably critical for the induction and regulation of Ab production (23). Dsg3-reactive Th1 and Th2 cells have been identified by several studies in PV patients.…”

Dichotomy of Autoreactive Th1 and Th2 Cell Responses to Desmoglein 3 in Patients with Pemphigus Vulgaris (PV) and Healthy Carriers of PV-Associated HLA Class II Alleles

“…A critical role of autoreactive T cells in the induction and regulation of Ab production has been suggested by a recent study (18). By use of ELISPOT assay, anti-Dsg3 autoAb secreted by autoreactive B cells were detected upon in vitro stimulation of peripheral lymphocytes from PV patients with Dsg3.…”

“…By use of ELISPOT assay, anti-Dsg3 autoAb secreted by autoreactive B cells were detected upon in vitro stimulation of peripheral lymphocytes from PV patients with Dsg3. In contrast, activation of autoreactive B cells was virtually absent upon depletion of the peripheral lymphocytes from CD4 ϩ cells (18). In addition, the adoptive in vivo transfer of splenocytes from Dsg3 Ϫ/Ϫ mice immunized with Dsg3 into Dsg3 ϩ/ϩ recombinase-activating gene 2 Ϫ/Ϫ mice led to the induction of Dsg3-specific autoAb accompanied by mucosal erosions characteristic for PV.…”